DSZD utilization in desulfurization of DBT by rhodococcus sp. IGTS8

The invention relates to the discovery that the rate of reaction of the desulfurization of fossil fuels is enhanced by the addition of an oxidoreductase to the biocatalyst. The invention is drawn to a method for enhancing the rate of desulfurizing a fossil fuel containing organic sulfur compounds, comprising the steps of:a) contacting the fossil fuel with an aqueous phase containing a biocatalyst capable of cleaving carbon-sulfur bonds and a rate-enhancing amount of an oxidoreductase, thereby forming a fossil fuel and aqueous phase mixture;b) maintaining the mixture of step (a) under conditions sufficient for cleavage of the carbon-sulfur bonds of the organic sulfur molecules by the biocatalyst, thereby resulting in a fossil fuel having a reduced organic sulfur content; andc) separating the fossil fuel having a reduced organic sulfur content from the resulting aqueous phase. The invention also relates to a recombinant microorganism containing one or more recombinant DNA molecules which encode a biocatalyst capable of desulfurizing a fossil fuel containing organic sulfur molecules and which encode an oxidoreductase. The invention also relates to a composition comprising (a) a biocatalyst capable of desulfurizing a fossil fuel containing organic sulfur molecules and (b) an oxidoreductase.

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Claims

1. An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes a biocatalyst which oxidatively desulfurizes a fossil fuel which contains organic sulfur molecules via nicotinamide adenine dinucleotide-dependent manner.

2. The DNA molecule of claim 1 wherein the group III alcohol dehydrogenase is N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase.

3. The DNA molecule of claim 1 wherein the group III alcohol dehydrogenase is of Rhodococcus origin.

4. The DNA molecule of claim 3 wherein the DNA molecule which encodes the biocatalyst is derived from Rhodococcus sp. ATCC 53968.

5. A microorganism containing a recombinant DNA molecule which encodes:

(a) a group III alcohol dehydrogenase; and
(b) one or more biodesulfurization enzymes which are components of a biocatalyst that oxidatively cleaves carbon-sulfur bonds of an organic sulfur molecule in a nicotinamide adenine dinucleotide-dependent manner.

6. The microorganism of claim 5 wherein the group III alcohol dehydrogenase is N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase.

7. The microorganism of claim 5 wherein the DNA which encodes the group III alcohol dehydrogenase is of Rhodococcus origin.

8. The microorganism of claim 7 wherein the DNA which encodes one or more biodesulfurization enzymes is derived from Rhodococcus sp. ATCC 53968.

9. The DNA molecule of claim 3 wherein the group III alcohol dehydrogenase is ThcE.

10. An isolated DNA molecule comprising DNA which encodes a group III alcohol dehydrogenase and DNA which encodes one or more biodesulfurization enzymes selected from the group consisting of DszA, DszB and DszC.

11. The DNA molecule of claim 10 wherein the group III alcohol dehydrogenase is an N,N'-dimethyl-4-nitrosoaniline-dependent alcohol oxidoreductase.

12. The DNA molecule of claim 10 wherein the group III alcohol dehydrogenase is of Rhodococcus origin.

13. The DNA molecule of claim 12 wherein the group III alcohol dehydrogenase is ThcE.

14. The microorganism of claim 5 wherein the biodesulfurization enzymes are selected from the group consisting of DszA, DszB and DszC.

15. The microorganism of claim 14 wherein the group III alcohol dehydrogenase is ThcE.

Referenced Cited
U.S. Patent Documents
5002888 March 26, 1991 Kilbane, II
5104801 April 14, 1992 Kilbane, II
5132219 July 21, 1992 Kilbane, II
5198341 March 30, 1993 Kilbane, II
5344778 September 6, 1994 Kilbane, II
5356801 October 18, 1994 Rambosek et al.
5356813 October 18, 1994 Monticello
5358870 October 25, 1994 Monticello et al.
Foreign Patent Documents
94/01563 January 1994 WOX
Other references
  • Monticello, Daniel, J. and Kilbane, John J., "Practical Considerations in Biodesulfurization of Petroleum," IGT's Third International Symposium on Gas, Oil, Coal and Environmental Biotechnology (Dec. 3-5 1990), New Orleans, LA. Denome, Sylvia A. et al., "Characterization of the Desulfurization Genes from Rhodococcus sp. Strain IGTS8,"Journal of Bacteriology 176(21):6707-6716 (1994). Lei, Benfang et al., "Vibrio harveyi NADPH-Flavin Oxidoreductase: Cloning, Sequencing and Overexpression of the Gene and Purification and Characterization of the Cloned Enzyme," Journal of Bacteriology 176 (12):3553-3558 (1994). Ohshiro, Takashi et al., "Enzymatic desulfurization of dibenzothiopene by a cell-free system of Rhodococcus erythropolis D-1," FEMS Microbiology Letters, 118:341-344 (1994). Ohshiro, Takashi et al., "Involvement of Flavin Coenzyme in Dibenzothiophene Degrading Enzyme System from Rhodococcus erythropolis D-1," Biosci, Biotech. Biochem., 59(7) :1349-1351 (1995). Piddington, Christopher S. et al., "Sequence and Molecular Characterization of a DNA Region Encoding the Dibenzothiophene Desulfurization Operon of Rhodococcus sp. Strain IGTS8,"Applied and Environmental Microbiology, 61(2):468-475 (1995). Nagy, Istvan et al., "Characterization of the Rhodococcus sp. NI86/21 gene encoding alcohol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase inducible by atrazine and thiocarbamate herbicides," Arch Microbiol. , 163:439-446 (1995). Parry, Ronald J. et al., "Cloning, Analysis, and Overexpression of the Gene Encoding Isobutylamine N-Hydroxylase from the Valanimycin Producer, Streptomyces viridifaciens," Journal of Bacteriology, 179(2):409-416 (1997). Arunachalam, Usha et al., "Mechanism of p-Hydroxyphenylacetate-3-hydroxylase," The Journal of Biological Chemistry, 269(1):150-155 (1994). Lei, Benfang and Tu, Shiao-Chun, "Gene Overexpression, Purification, and Identification of a Desulfurization Enzyme from Rhodococcus sp. Strain IGTS8 as a Sulfide/Sulfoxide Monooxygenase," Journal of Bacteriology, 178(19):5699-5705 (1996). Arunachalam, Usha et al., "p-Hydroxyphenylacetate-3-hydroxylase," The Journal of Biological Chemistry, 267(36):25848-25855 (1992). Kendrew, Steven G. et al., "Identification of a Flavin:NADH Oxidoreductase Involved in the Biosynthesis of Actinorhodin," The Journal of Biological Chemistry, 270(29):17339-17343 (1995). Thibaut, Dennis et al., "Purification of the Two-Enzyme System Catalyzing the Oxidation of the D-Proline Residue of Pristinamycin II.sub.B during the Last Step of Pristinamycin II.sub.A Biosynthesis," Journal of Bacteriology, 177(18):5199-5205 (1995). Blanc, Veronique et al., "Cloning and Analysis of Structural Genes from Streptomyces pristinaespiralis Encoding Enzymes Involved in the Conversion of Pristinamycin II.sub.B to Pristinamycin II.sub.A (PII.sub.A): PII.sub.A Synthase and NADH:Riboflavin 5'-Phosphate Oxidoreductase," Journal of Bacteriology, 177(18):5206-5214 (1995). Knobel, Hans-Rudolf et al., "Cloning and Characterization of the Genes Encoding Nitrilotriacetate Monooxygenase of Chelatobacter heintzii ATCC 29600," Journal of Bacteriology, 178(21):6123-6132 (1996). Uetz, Thomas et al., "Purification and Characterization of a Two-Component Monooxygenase That Hydroxylates Nitrilotriacetate from Chelatobacter Strain ATCC 29600," Journal of Bacteriology, 174(4):1179-118 (1992). Fernandez-Moreno, Miguel A. et al., "Nucleotide Sequence and Deduced Functions of a Set of Cotranscribed Genes of Streptomyces coelicolor A3 (2) Including the Polyketide Synthase for the Antibiotic Actinorhodin," The Journal of Biological Chemistry, 267(27):19278-19290 (1992). Prieto, Maria A. and Garcia, Jose L., "Molecular Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase of Escherichia coli," The Journal of Biological Chemistry, 269 (36) :22823-22829 (1994). Gray, Kevin A. et al., "Molecular mechanisms of biocatalytic desulfurization of fossil fuels," Nature Biotechnology, 14:1705-1709 (1996). Xi, L et al., "A Flavin Reductase Stimulates DszA and DszC Proteins of Rhodococcus erythropolis IGTS8 in Vitro" Biochemical and Biophysical Research Communications, 230:73-75 (1997).
Patent History
Patent number: 5811285
Type: Grant
Filed: Sep 19, 1996
Date of Patent: Sep 22, 1998
Assignee: Energy BioSystems Corporation (The Woodlands, TX)
Inventors: Kevin A. Gray (The Woodlands, TX), Charles H. Squires (The Woodlands, TX), Daniel J. Monticello (The Woodlands, TX)
Primary Examiner: Rebecca E. Prouty
Assistant Examiner: Einar Stole
Law Firm: Hamilton, Brooks, Smith & Reynolds, P.C.
Application Number: 8/715,554
Classifications
Current U.S. Class: 435/2523; 435/2523; 435/3201; 536/233; 935/22
International Classification: C12N 120; C12N 1500; C07H 2104;